Substituted phenylglycylcephalosporins

ABSTRACT

Cephalosporin compounds having a substituted phenylglycyl substituent at the 7-position and any of a variety of groups at the 3-position are prepared by acylation of a 7-aminocephalosporanic acid. The compounds have antibacterial activity.

This is a division of application Ser. No. 384,771 filed Aug. 1, 1973,now U.S. Pat. No. 3,953,439.

This invention relates to cephalosporia compounds having antibacterialactivity. In particular, this invention relates to certain compoundshaving a substituted phenyl glycyl substituent at position 7 of thecephem nucleus.

The compounds of this invention are represented by the followingstructural formula: ##STR1## in which: Y is O, NH or S;

n is one to five;

R is NH₂ or OR', where R' is hydrogen or lower alkyl of from one to fourcarbon atoms;

M is hydrogen or an alkali metal or ammonium cation;

A is hydrogen, acetoxy, pyridyl, SHet, SR' or OR', where R' is hydrogenor alkyl of from one to four carbon atoms; and

Het is a five or six-membered heterocyclic group containing carbon andone to four atoms selected from the group consisting of N, O and S, eachsuch group being unsubstituted or substituted with from one to twogroups selected from lower alkyl and alkoxyalkyl, each alkoxy or alkylhaving from one to four carbon atoms, hydroxy, trifluoromethyl, and SR',where R' is hydrogen or alkyl of from one to four carbon atoms.

Preferred compounds within the scope of the formula I are those where nis one or two and A is hydrogen, acetoxy or SHet. Especially preferredare those where Het is unsubstituted or methyl substituted1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3,4-tetrazolyl, oxazolyl,thiazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, or 1,2,4-thiadiazolyl.

It will be recognized that when the substituent on the heterocyclicgroup is hydroxy or mercapto, it is possible for the substituent toexist in either of two tautomeric forms, i.e. the oxo or thiono form.The compounds may exist exclusively as one of the two tautomers or maybe in equilibrium between the two; however, these are all includedwithin the scope of this invention and the use of one structure or nameis intended to indicate both tautomers and mixture thereof.

Cephalosporin derivatives substituted in the 7-position with aphenylglycylacetamido side chain are well documented in the prior art.Amino, alkoxy, and alkylmercapto substitution on the phenyl moiety isdescribed in Belgian Patent 780,021 and Irish Patent 296/64, while loweralklanoylaminophenylglycyl substituents are described in U.S. Pat. No.3,634,418 and 3,464,985. Also disclosed in U.S. Pat. No. 3,634,418, aswell as in Irish Patent 296/64, are phenylglycyl groups substituted withvarious acyloxy groups. Carboxyalkylphenyl and carbamoylphenylsubstituents are described in Netherlands Patent 69,02013 and IrishPatent 296/64. Netherlands Patent 69,02013 also disclosesphenylglycylcephalosporins with a phenyl group substituted with acarboxamidomethyl group.

Substitution by an S-heterocyclicthiomethyl group at the 3-position ofthe cephem nucleus is also known in the prior art and is disclosed in,among other patents, U.S. Pat. No. 3,641,021 where Het ismethylthiadiazolyl or methyltetrazolyl and Japanese Patent 7124400 whereHet is alkyl substituted thiazolyl and tetrazolyl. Although the two lastcited patents describe cephalosporins also having aphenylglycylacetamido substituent at the 7-position, the phenyl groupis, in both cases, either unsubstituted or substituted with lower alkyl,lower alkoxy, hydroxy, amino, nitro or halogen. U.S. Pat. Nos. 3,516,997and 3,530,123 also describe 3-S-heterocyclic compounds but withdifferent 7 substituents.

The compounds of this invention are prepared by acylation of a7-aminocephalosporanic acid with an appropriately substitutedphenylglycine. Prior to acylation it is desirable to protect the aminogroup of the glycine moiety with an easily removable protective groupsuch as t-butoxycarbonyl, benzyloxycarbonyl, trichloroethoxycarbonyl, orsimilar protective group commonly used in the synthesis of peptides. Inmost cases, the carboxyl group is activated prior to or during theacylation reaction by conversion to the 2,4-dinitrophenyl orN-hydroxysuccinimidyl ester. If an ester of the carboxyl group on thecephalosporin nucleus is used as an acylation substrate, e.g., abenzhydryl, t-butyl, trichloroethyl or a benzyl ester, theamine-protected phenylglycine can be coupled directly to the 7-aminogroup by using a carbodiimide such as dicyclohexylcarbodiimide.Alternatively, the protected phenylglycine can be activated forcondensation by reacting it first with carbonyldiimidazole or itsequivalent.

Following the acylation, the protective groups can be removed with anacid such as trifluoroacetic acid. The resulting salt is converted tothe zwitterionic product by means of a basic ion exchange resin such aspolystyreneamine ion exchange resin. (Amberlite IR-45) or else bybasification of an aqueous solution of the salt.

The compounds are also prepared by displacement of a 7-acylated3-acetoxymethylcephalosporin with a mercaptoheterocycle is an aqueous,slightly basic medium.

The suitably blocked substituted phenylglycines are prepared fromreaction of an appropriately substituted α-aminophenylacetic acid suchas p-amino-D-N-acetylphenylglycine (U.S. Pat. No. 3,479,339), and anω-haloalkanoic acid or acetamide derivative such as α-bromoacetic acidt-butyl ester.

The 7-aminocephalosporins where A is SHet are prepared by known methodsfrom 7-aminocephalosporanic acid (7-ACA) and the appropriateheterocyclic mercapto compound. The compounds where A is hydrogen oracetoxy are prepared by previously known methods from materials known inthe art.

The compounds of this invention have antibacterial activity againstGram-positive and Gram-negative organisms. Minimum inhibitoryconcentrations (MIC's) ranged from 0.4 to >200 μg/ml in in vitrotesting. Upon in vivo subcutaneous and oral administration the compoundsalso showed significant activity. In addition, many of the compoundsshowed activity against Proteus morgani, an indole-positive bacterium,against which most known cephalosporins and penicillins are inactive.These results are summarized in Tables 1 and 2; compound numberscorresponding to structures are given in the experimental section.

These compounds are formulated and administered by injection in the samemanner as other cephalosporins in dosages of from 250 to 1000 mg. Dosageis dependent upon the age and weight of the subject and on the infectionbeing treated and can be determined by those skilled in the art based onthe data disclosed herein and experience with known cephalosporins.

                                      TABLE 1                                     __________________________________________________________________________    In Vitro Activity (MIC)                                                       __________________________________________________________________________                                                Sal.                                  S.   S.        Strep.                   para      Entero.                                                                            Proteus            Com-                                                                              aureus                                                                             aureus                                                                             S.   faecalis                                                                           E. coli                                                                            E. coli                                                                            Kleb.                                                                              Kleb.                                                                              typhi                                                                              Shig.                                                                              aerog.                                                                             morg-              pound                                                                             HH   SK   aureus                                                                             HH   SK   HH   pneumo.                                                                            pneumo.                                                                            ATCC paradys                                                                            ATCC ani                No. 127  23390                                                                              Villaluz                                                                           34358                                                                              12140                                                                              33779                                                                              SK 4200                                                                            SK 1200                                                                            12176                                                                              HH 112                                                                             13048                                                                              179                __________________________________________________________________________    48536                                                                             12.5 12.5 100  100  3.1  6.3  1.6  3.1  1.6  3.1  12.5 2.5                51436                                                                             12.5 12.5 200  200  25   25   12.5 50   12.5 25   100  --                 99316                                                                             12.5 12.5 100  >200 12.5 50   12.5 6.3  6.3  12.5 50   >200               03616                                                                             6.3  6.3  25   100  6.3  12.5 1.6  3.1  25   25   25   200                70326                                                                             6.3,12.5                                                                           6.3,12.5                                                                           50,100                                                                             100,200                                                                            0.8,1.6                                                                            1.6,3.1                                                                            0.4,0.8                                                                            0.8,0.8                                                                            0.4,0.8                                                                            0.4,0.8                                                                            1.6,3.1                                                                            6.3                04726                                                                             12.5 12.5 100  200  25   12.5 3.1  3.1  3.1  6.3  25   --                 61726                                                                             6.3  6.3  50   50   12.5 25   6.3  6.3  12.5 6.3  25   200                86526                                                                             6.3  3.1  25   50   25   25   12.5 6.3  6.3  6.3  100  --                 60326                                                                             12.5 12.5 200  200  6.3  25   6.3  6.3  3.1  6.3  25   >200               *08016                                                                            50   50   >200 >200 50   50   --   25   25   50   100  --                 59216                                                                             6.3  25   100  100  6.3  12.5 3.1  6.3  6.3  6.3  12.5 200                *59216                                                                            6.3-25                                                                             100-200                                                                            100-200                                                                            50-200                                                                             3.1-12.5                                                                           6.3-25                                                                             1.6-6.3                                                                            3.1-12.5                                                                           3.1-12.5                                                                           1.6-12.5                                                                           12.5-50                                                                            --                 03326                                                                             12.5 100  200  200  6.3  25   6.3  3.1  3.1  6.3  25   200                42526                                                                             25   25   200  >200 6.3  12.5 3.1  3.1  6.3  6.3  12.5 100                07826                                                                             6.3  6.3  200  100  100  >200 100  100  50   50   >200 --                 26436                                                                             6.3  6.3  25   50   12.5 12.5 6.3  6.3  3.1  6.3  25   50                 19736                                                                             12.5 12.5 200  100  50   100  50   50   25   25   >200 >200               96826                                                                             3.1  3.1  25   25   25   50   25   25   25   12.5 50   25                 00036                                                                             3.1  3.1  200  50   25   50   25   25   25   12.5 100  --                 71816                                                                             3.1,12.5                                                                           1.6,3.1                                                                            25   25   6.3  12.5 3.1  6.3,1.6                                                                            3.1,3.1                                                                            6.3,3.1                                                                            12.5,6.3                                                                           100,                                                                          200                *71816                                                                            25   12.5 100  200  50   50   25   50   12.5 12.5 100  --                 32626                                                                             12.5 6.3  50   200  12.5 25   6.3  12.5 12.5 6.3  12.5 100                91726                                                                             6.3  6.3  50   200  50   25   12.5 12.5 6.3  6.3  50   --                 96146                                                                             6.3  6.3  50   25   50   100  50   50   25   25   100  >200               72546                                                                             12.5 12.5 50   50   25   50   12.5 25   12.5 12.5 50   >200               76746                                                                             6.3  6.3  50   100  1.6  1.6  0.8  1.6  3.1  3.1  1.6  6.3                __________________________________________________________________________     *Compound with DL side chain, all others D                               

                                      TABLE 2                                     __________________________________________________________________________    In Vivo Activity                                                              __________________________________________________________________________    Compound                                                                            ED.sub.50 (mg/kg)                                                       No.   E.coli 12140 Klebs.pneumo.4200                                          __________________________________________________________________________    s.c.        p.o.   s.c.   p.o.                                                __________________________________________________________________________    99316 25,37 42,25  29,11  15.5,15.5                                           03616 6.2   14.5   11.5   16                                                  70326 2.2   11     1.7, <3                                                                              4,3                                                 04726 12.5  46     9.5    14.5                                                61726 --    --     50     >50                                                 86526 18    42     7.2    10                                                  60326 11.2  200    12.5   63                                                  59216 9.5   25     4.8    21.5                                                *59216                                                                              6.2,9,11                                                                            50,21.5,40                                                                           7.2,3,6.2                                                                            29,22.5,17.6                                        03326 8.4   25     4.6    17.7                                                42526 9.5   >50    5.4    33                                                  26436 1.2   9.6    1.5    5.5                                                 19736 34    25     --     --                                                  96826 <3    9.4    7.2    12.5                                                71816 4.5   12.5   7.5    6.2                                                 *71816                                                                              13.7  21.5   11.5   13.7                                                32626 8.7   >50    15.5   >50                                                 91726 14    25     12.5   13.6                                                48536 2.0   6.4    7.8    12.5                                                96146 4.7   16     2.9    17                                                  __________________________________________________________________________     *Compound with DL side chain; all others D                               

It will be recognized that, due to the asymmetric α-carbon atom in the7-acetamido group, optical isomers will exist. Racemic or resolvedproducts are obtained, depending upon whether a racemic or resolvedphenylglycine side chain is used or upon the reaction conditions used toform the acylated cephalosporin. The resolved side chain acids arereadily obtained from the racemic compounds by resolution according towell known methods including fractional crystallization of a salt formedwith an optically active acid or base. Both the resolved and racemicproducts obtained from the D & DL-side chains are within the scope ofthis invention.

Due to the presence of both an amine group and a carboxylic acid groupin the cephalosporin compounds of this invention, it is possible, bystandard methods, to prepare both acid and base salts ofpharmaceutically acceptable nontoxic acids and bases as well as thezwitterionic forms of the compounds. Salts, when obtained, are readilyconverted to the zwitterions by known methods. It is to be understoodthat these salts are included within the scope of this invention.

This invention also consists of the novel intermediate compoundsrepresented by the structural formula: ##STR2## in which: Y is O, NH orS;

n is one to five;

R is NH₂ or OR', where R' is hydrogen or lower alkyl of from one to fourcarbon atoms; and

R² is hydrogen or t-butoxycarbonyl.

These intermediate compounds are prepared by condensing an N-protectedp-hydroxy, p-mercapto, or p-aminophenylglycine ester with aw-haloalkannic ester or amide. Removal of protective groups is bystandard methods.

The following examples illustrate the invention but are not to beconstrued as limiting the scope thereof. Temperatures are in degreesCentigrade unless otherwise stated. Each of the products orintermediates obtained as a trifluoroacetate salt is converted to itszwitterion by means of the procedure described in Example 6.

EXAMPLE 1 D-N-t-Butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine

p-Amino-D-N-acetylphenylglycine (20.0 g, 0.095 mol) was slowly added to110 ml (1.4 mol) of trifluoroacetic acid at 5°. The reaction mixture wasstirred for 25 minutes, then cooled to -5° and diazotized with asolution of 4.65 g (.095 mol) of sodium nitrate in 14.5 ml of H₂ O. Thediazonium solution was added to a cold aqueous solution of 58.0 g (1.65mol) of sodium hydroxide, 28.4 g of sodium sulfide and 3.6 g of sulfur,then left to sit overnight at 25°. Acidification of the reaction mixtureto ph2 with conc.HCl gave a gum which was separated, combined with theresidue from extraction of the aqueous solution with butanol, anddissolved in 5% sodium bicarbonate. This solution was filtered throughCelite to remove excess sulfur, then oxidized with iodine and potassiumiodide. Extraction with sodium bisulfite, followed by acidification,gave the crude disulfide, which was recrystallized from methanol-ether(13.8 g, 65%). The product was refluxed in 30 ml of 1:1 dioxane --5% HClfor 14 hours to remove the acetyl protective group. Concentration andcrystallization from ethanol-ether gave 11.5 g (63%) amino acidhydrochloride salt. The product (4.37 g, 0.01 mol) was heated at 40°-60°for eight hours with 8.6 g (0.06 mol) of t-butoxycarbonyl azide and 3.2g (0.8 mol) of magnesium oxide. The reaction mixture was filtered,washed with 5% sodium bicarbonate and the combined filtrate and washingswere then extracted with ethyl acetate, acidified to pH2 andre-extracted with ethyl acetate. The extracts were washed with water andsaturated NaCl solution; dried (MgSO₄) and concentrated in vacuo to givea residue which was recrystallized from methylene chloride-hexane (3.5g, 63%).

The above disulfide (1.5 g, 2.7 mmol) was dissolved in 10 mls of acetoneand treated with 2.0 g (5.4 mmol) of tri-n-octylphosphine and 10 ml of10% methanol in water. The reaction mixture was stirred at 25° for onehour, then it was diluted with 5% sodium bicarbonate solution andextracted with ethyl acetate. The aqueous phase was acidified, extractedwith methylene chloride and the extracts were washed with water andsaturated NaCl solution, dried (Na₂ SO₄) and concentrated to dryness.The thiol (1 g, 3.5 mmol) was dissolved in 10 ml of ethanol and to thissolution was added 1 g (5.1 mmol) of α-bromoacetic acid t-butyl esterand 2.5 g (19 mmol) of diisopropylethylamine. The reaction mixture wasstirred for one hour, then extracted with dilute HCl in ethyl acetate.The organic extracts were dried (Na₂ SO₄) and concentrated to drynessand the residue was chromatographed on silica gel with 99:1chloroform:acetic acid to give 0.62 g ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine.

A solution of 0.5 g ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine in 10 mlof anisole or tetrahydrofuran is treated with trifluoroacetic acid at 0°and stirred for 1.5 hours. The reaction mixture is concentrated in vacuoand the residue triturated with ether to give theD-p-carboxymethylthiophenylglycine salt.

EXAMPLE 2 7-[D-α-Amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-desacetoxycephalosporanic acid (Compound No. 99316)

A mixture of 0.520 g (1.3 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine, 0.425 g(1.9 mmol) of 7-amino -3-desacetoxycephalosporanic acid and 0.330 g (1.6mmol) of dicyclohexylcarbodimide in 5 ml of methylene chloride wasstirred for three hours at 0°. The solvent was removed in vacuo and theresidue was shaken with ethyl acetate and 2.5% sulfuric acid. The layerswere separated and the organic phase was washed with 5% sodiumbicarbonate, water and sodium chloride solution, then dried (MgSO₄).Concentration yielded a residue that was dried in vacuo andchromatographed in silica gel with chloroform to give 0.505 g ofproduct. The acid was dissolved in 10 ml of a cold 10% solution ofanisole in trifluoroacetic acid and stirred at 0° for 1.25 hours.Concentration in vacuo gave a residue which was triturated with etherand redissolved in a minimum amount of ethanol. Addition of ether to theethanol solution caused precipitation of the salt.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.18 H.sub.19 N.sub.3 O.sub.6 S.sub.2.1/2CF.sub.3                         COOH.1/2 H.sub.2 O                                                            (503.529                                                        ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             45.32         45.66                                             H             4.00          4.30                                              N             8.35          8.42                                              ______________________________________                                    

The salt can be converted to the zwitterionic product by means of apolystyrene-amine ion exchange resin.

EXAMPLE 3 7-Amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid

To a suspension of 27.2 g (0.1 mol) of 7-ACA in 200 ml of water and 100ml of acetone was added a solution of 18.9 g of sodium bicarbonate in200 ml of water. The resultant solution was warmed on a steam bath and asolution of 14.5 g (0.125 mol) of 1-methyl-5-mercapto-1,2,3,4-tetrazolein 200 ml of acetone was added. The reaction mixture was refluxed for3.5 hours while maintaining the pH at 7.4-8.0 by addition of 5% sodiumbicarbonate solution. Acidification of the cooled reaction mixture to pH3.5 with 6N hydrochloric acid resulted in precipitation of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid which was collected, washed (H₂ O), and air dried (16 g, 49%).

EXAMPLE 47-Amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound was obtained from reaction of 7-ACA and5-methyl-2-mercapto-1,3,4-thiadiazole by the same procedure as describedin Example 3.

EXAMPLE 5 7-Amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound was obtained from reaction of 7-ACA and4-mercapto-1,2,3-triazole by the same procedure as described in Example3.

EXAMPLE 67-[D-α-Amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 03616)

A solution of 0.200 g (0.51 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine, 0.214 g(0.53 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, and 0.112 g (0.54 mmol) of dicyclohexylcarbodiimidein 10 ml of methylene chloride was stirred at 0° for one hour. Thereaction mixture was filtered and the filtrate was washed with 2.5%sulfuric acid, 5% sodium bicarbonate and water, dried (MgSO₄) andconcentrated to give crude7-[D-α-t-butoxycarbonylamino-α-(4'-carboxymethylthiophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid, di-t-butyl ester. The ester was dissolved in 10 ml of a 20%solution of anisole in trifluoroacetic acid and stirred at 0° for 1.5hours. The solvent was removed in vacuo and the residue was trituratedwith ether. The precipitated trifluoroacetate salt of the title compoundwas collected, dried in vacuo and recrystallized from ethanol-ether(0.191 g).

    ______________________________________                                        Analysis of salt:                                                                           C.sub.21 H.sub.21 N.sub.5 O.sub.6 S.sub.4.1/2 CF.sub.3                        COOH.1/2 H.sub.2 O                                                            (633.726)                                                       ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             41.71         41.74                                             H              3.58          3.79                                             N             11.05         10.76                                             ______________________________________                                    

The trifluoroacetate salt is converted to the zwitterion by stirring anaqueous solution of the salt with a polystyrene-amine ion-exchange resin(Amberlite IR-45) for one hour at 25°. The resin is then filtered offand the aqueous solution is lyophilized to yield the zwitterioniccephalosporin which may be converted to the sodium salt by addition of a30% solution of sodium 2-ethylhexanoate in isopropanol.

EXAMPLE 77-[D-α-Amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 70326)

The title compound was obtained from reaction of 0.5 g (1.28 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine, 0.5 g(1.3 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, and 0.265 g (1.3 mmol) of dicyclohexylcarbodiimideby the same procedures as described in Example 6.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.20 H.sub.21 N.sub.7 O.sub.6 S.sub.3.1 H.sub.2 O                         (569.658)                                                       ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             42.17         42.29                                             H              4.06          4.13                                             N             17.21         15.41                                             ______________________________________                                    

EXAMPLE 87-[D-α-Amino-α-(4'-carboxymethylthiophenyl)-acetamido]-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 04726)

A mixture of 1.12 g (2.82 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine, 0.324 g(2.82 mmol) of N-hydroxysuccinimide, and 0.58 g (2.82 mmol) ofdicyclohexylcarbodiimide in 20 ml of acetonitrile was stirred at 25° for12 hours. The reaction mixture was filtered and concentrated to dryness.The residue was then dissolved in ether and the ethereal solution wasfiltered and concentrated to yield the activated ester as a foam.

A solution of 0.88 g (2.82 mmol) of7-amino-3(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylic acid and0.57 g (5.64 mmol) of triethylamine in 20 ml of dry pyridine was stirredfor 0.5 hour. The activated ester from above was then added and thereaction mixture was stirred at 25° for 4.5 hours. Addition of ether tothe reaction mixture caused precipitation of the salt which wascollected by filtration and dissolved in water. Ethyl acetate was addedto the aqueous solution and the mixture was acidified with dilute HCland filtered. The layers were separated and the organic layer was washed(H₂ O), treated with activated carbon, dried (MgSO₄) and concentrated invacuo to yield 0.450 g (23%) of product. The deblocked acid was obtainedby stirring the t-butyl ester derivative with a solution of 1 ml ofanisole in 5 ml of trifluoroacetic acid for two hours at 0° followed byconcentration in vacuo. Crystallization occurred upon trituration withether.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.20 H.sub.20 N.sub.6 O.sub.6 S.sub.3 .CF.sub.3 COOH.                     1/4(CH.sub.3 CH.sub.2).sub.2 O (669.187)                        ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             41.28         41.68                                             H              3.54          3.72                                             N             12.56         13.09                                             ______________________________________                                    

The zwitterionic product is obtained as described in Example 6.

EXAMPLE 9 D-N-t-Butoxycarbonyl-p-t-butoxycarbonylethylthiophenylglycine

The title compound was obtained from reaction ofp-amino-D-N-acetylphenylglycine, 3-bromopropionic acid t-butyl ester anddiisopropylamine by the same procedure as described in Example 1.

D-p-carboxyethylthiophenylglycine is prepared as its salts by treatingthe title compound with trifluoroacetic acid as described in Example 1.

EXAMPLE 107-[D-α-Amino-α-(4'-carboxyethylthiophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 61726)

The title compound was obtained from reaction of 0.135 g (0.33 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylethylthiophenylglycine, 0.110 g(0.28 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, and 0.060 g (0.28 mmol) of dicyclohexylcarbodiimideby the same procedure as described in Example 6. Deblocking wasaccomplished by stirring the crude product with 0.5 ml of anisole and 5ml of trifluoroacetic acid at 0° for one hour, then at 15° for 0.5 hour.The acid (0.105 g; 65%) was precipitated as its salt by trituration ofthe residue with ether.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.22 H.sub.23 N.sub.5 O.sub.6 S.sub.4.1 H.sub.2 O                         (599.746)                                                       ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             44.06         43.72                                             H              4.20          3.89                                             N             11.67         11.45                                             ______________________________________                                    

EXAMPLE 11 D-N-t-butoxycarbonyl-p-carbamylmethylthiophenylglycine

A mixture of 1.0 g (3.7 mmol) ofD-N-t-butoxycarbonyl-p-mercaptophenylglycine and 0.4 g (4.3 mmol) ofα-chloro-acetamide in 10 ml of ethanol was stirred under nitrogen with1.0 ml of triethylamine for four hours. The reaction mixture was thendiluted with water, acidified with dilute HCl and extracted with ethylacetate. The organic phase was shaken with 5% sodium carbonate and themixture was acidified then extracted with ethyl acetate. The extractswere washed with saturated sodium chloride solution, dried (Na₂ SO₄),and concentrated to give a residue which was crystallized from ethanol(0.830 g; 66%).

D-p-carbamylmethylthiophenylglycine is obtained as its salt fromtreatment of the title compound with trifluoroacetic acid as describedin Example 1.

EXAMPLE 127-[D-α-Amino-α-(4'-carbamylmethylthiophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 86526)

The title compound was obtained from 0.175 g (0.52 mmol) ofD-N-t-butoxycarbonyl-p-carbamylmethylthiophenylglycine, 0.200 g (0.50mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, and 0.120 g (0.58 mmol) of dicyclohexylcarbodiimideby the same procedure as described in Example 6. Before deblocking, thecrude intermediate was purified by chromatography on silica gel withchloroform-ethyl acetate.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.21 H.sub.22 N.sub.6 O.sub.5 S.sub.4. CF.sub.3                           CO.sub.2 H                                                      ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             40.58         41.25                                             H              3.41          3.72                                             N             12.35         12.85                                             ______________________________________                                    

The zwitterionic product is obtained as in Example 6.

EXAMPLE 13 D-N-t-Butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine

A solution of 25 g (0.129 mol) of diphenyldiazomethane in 210 ml of drybenzene was added dropwise to a stirred solution of 26.7 g (0.100 mol)of D-N-t-butoxycarbonyl-p-hydroxyphenylglycine in 200 ml of dry benzeneand 30 ml of dry THF. After stirring at 25° for 4.5 hours, the reactionmixture was concentrated and the residue was dissolved in ether. Theethereal solution was washed with 5% sodium bicarbonate and with water,dried and concentrated in vacuo to yield a residue which was dissolvedin a small volume of chloroform and precipitated by addition of pet.ether to give 34.8 g (80%) ofD-N-t-butoxycarbonyl-p-hydroxyphenylglycine benzhydryl ester.

The benzhydryl ester (4.33 g; 0.01 mol) was stirred at 25° for 1.5 hourswith 4 ml of α-bromoacetic acid t-butyl ester in 40 ml of drydimethylformamide. The reaction mixture was diluted with water andextracted with ether. Concentration gave an oily residue which washeated (90°) in vacuo, then dissolved in 150 ml of ethanol andhydrogenolyzed over 2 g of 10% palladium on carbon to give the titlecompound (3.2 g; 84%).

D-p-carboxymethoxyphenylglycine is obtained as its salt from treatmentof the title compound with trifluoroacetic acid as described in Example1.

EXAMPLE 147-[D-α-Amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 59216)

The title compound was obtained from 0.87 g (2.28 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine, 0.92 g (2.3mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.475 g (2.3 mmol) of dicyclohexylcarbodiimide bythe same procedure as described in Example 6. The intermediate waspurified by chromatography on silica gel with chloroform beforedeblocking.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.21 H.sub.21 N.sub.5 O.sub.7 S.sub.3.2H.sub.2 O.                         0.2(C.sub.2 H.sub.5).sub.2 O (602.494)                          ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             43.46         43.93                                             H              4.52          4.23                                             N             11.63         11.06                                             ______________________________________                                    

EXAMPLE 157-[DL-α-amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid (Compound No. 08016)

The title compound was obtained from 0.50 g (1.31 mmol) ofDL-N-t-butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine, 0.372 g(1.38 mmol) of 7-amino-3-desacetoxycephalosporanic acid t-butyl esterand 0.27 g (1.31 mmol) of dicyclohexylcarbodiimide by the same procedureas described in Example 6.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.18 H.sub.19 N.sub.3 O.sub.7 S.11/2H.sub.2 O                             (448.464)                                                       ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             48.21         48.45                                             H             4.94          4.92                                              N             9.37          9.12                                              ______________________________________                                    

EXAMPLE 167-[D-α-Amino-α-(4'-carboxymethoxyphenyl)acetamido]-cephalosporanic acid(Compound No. 60326)

The title compound was obtained from 0.656 g (0.002 mol) of 7-ACAt-butyl ester, 0.762 g (0.002 mol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine, and 0.412 g(0.002 mol) of dicyclohexylcarbodiimide by the same procedure asdescribed in Example 6.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.20 H.sub.21 N.sub.3 O.sub.9 S.O.4 CF.sub.3 COOH.1.25                    H.sub.2 O                                                                     (547.610)                                                       ______________________________________                                                    Theory      found                                                 ______________________________________                                        C             45.62         45.77                                             H             4.40          4.22                                              N             7.67          7.42                                              F             4.16          4.22                                              ______________________________________                                    

The zwitterionic product is obtained as described in Example 6.

EXAMPLE 177-[D-α-Amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 03326)

The title compound was obtained from 0.500 g (1.6 mmol) of7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylic acid and0.780 g (1.6 mmol) of the activated ester ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine by the sameprocedure as described in Example 8.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.20 H.sub.20 N.sub.6 C.sub.7 S.sub.2.1.75 H.sub.2 O                      (552.088)                                                       ______________________________________                                                    Theory      Found                                                 ______________________________________                                        C             43.51         44.04                                             H              4.29          4.19                                             N             15.22         13.57                                             ______________________________________                                    

EXAMPLE 187-[D-α-Amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 42526)

The title compound was obtained from 0.84 g (2.55 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and 1.22 g (2.55 mmol) of the activated ester ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethoxyphenylglycine by the sameprocedure as described in Example 8.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.20 H.sub.21 N.sub.7 O.sub.7 S.sub.2 . 1 H.sub.2 O                       (553.592)                                                       ______________________________________                                                    Theory    Found                                                   ______________________________________                                        C             43.39       43.28                                               H              4.19        4.10                                               N             17.71       15.77                                               ______________________________________                                    

EXAMPLE 19 D-N-t-Butoxycarbonyl-p-carbamylmethoxyphenylglycine

A mixture of 0.433 g (1.0 mmol) ofD-N-t-butoxycarbonyl-p-hydroxyphenylglycine benzhydryl ester, 0.555 g(3.0 mmol) of α-iodoacetamide and 0.414 g (3.0 mmol) of potassiumcarbonate in 10 ml of dry dimethylformamide was stirred at 25° for 12hours. The reaction mixture was then diluted with water and extractedtwice with ethyl acetate. The extracts were washed (H₂ O), dried (NaSO₄)and concentrated in vacuo to give 0.4 g (81.5%) of a residue whichcrystallized when dissolved in ether and triturated with pet. ether.

The product from above (3.9 g; 7.35 mmol) was dissolved in 50 ml ofglacial acetic acid and hydrogenolyzed over 1.0 g of 10% palladium oncarbon to give 1.7 g (66%) of the title compound.

D-p-carbamymethoxyphenylglycine is obtained as its salt from treatmentof the title compound with trifluoroacetic acid as described in Example1.

EXAMPLE 207-[D-α-Amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid (Compound No. 00036)

The title compound was obtained from 0.81 g (2.5 mmol) ofD-N-t-butoxycarbonyl-p-carbamylmethoxyphenylglycine, 0.675 g (2.5 mmol)of 7-amino-3-desacetoxycephalosporanic acid t-butyl ester and 0.515 g(2.5 mmol) of dicyclohexylcarbodiimide by the same procedure asdescribed in Example 6. Before deblocking, the crude intermediate waspurified by chromatography on silica gel.

    ______________________________________                                        Analysis of Salt:                                                                           C.sub.18 H.sub.20 N.sub.4 O.sub.6 S . 1/2CF.sub.3 COOH .                      3/4 H.sub.2 O                                                                 (490.983)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          46.48          46.51                                               H           4.52           4.59                                               N          11.41          11.29                                               ______________________________________                                    

The zwitterionic product is obtained as described in Example 6.

EXAMPLE 217-[D-α-Amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 71816)

The title compound was obtained from 0.648 g (2.0 mmol) ofD-N-t-butoxycarbonyl-p-carbamylmethoxyphenylglycine, 0.800 g (2.0 mmol)of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.412 g (2.0 mmol) of dicyclohexylcarbodiimide bythe same procedure as described in Example 6.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.21 H.sub.22 N.sub.6 O.sub.6 S.sub.3 . 1 H.sub.2 O .                     1/4                                                                           (C.sub.2 H.sub.5).sub.2 O (587.200)                             ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          45.00          45.13                                               H           4.55           4.54                                               N          14.31          13.90                                               ______________________________________                                    

EXAMPLE 22 7-[D-α-Amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 91726)

The title compound was obtained from 0.855 g (2.73 mmol) of7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylic acid and1.15 g (2.73 mmol) of the activated ester ofD-N-t-butoxycarbonyl-p-carbamylmethoxyphenylglycine by the sameprocedure as described in Example 8.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.20 H.sub.21 N.sub.7 O.sub.6 S.sub.2 . CF.sub.3 COOH .                   1 H.sub.2 O . 1/2                                                             (C.sub.2 H.sub.5).sub.2 O (688.684)                             ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          41.86          42.20                                               H           4.24           3.97                                               N          14.24          13.71                                               F           8.28           8.06                                               ______________________________________                                    

The zwitterionic product is obtained as described in Example 6.

EXAMPLE 237-[D-α-Amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 32626)

The title compound was obtained from 0.985 g (3.0 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and 1.30 g (3.1 mmol) of the activated ester ofD-N-t-butoxycarbonyl-p-carbamylmethoxyphenylglycine by the sameprocedure as described in Example 8.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.20 H.sub.22 N.sub.8 O.sub.6 S.sub.2 . 1/2 H.sub.2 O                     (543.600)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          44.19          44.26                                               H           4.26           4.33                                               N          20.62          17.44                                               ______________________________________                                    

EXAMPLE 24 D-N-t-Butoxycarbonyl-p-carbomethoxymethoxyphenylglycine

The title compound was obtained from 4.33 g (0.01 mol) ofD-N-t-butoxycarbonyl-p-hydroxyphenylglycine benzhydryl ester, 4 ml ofα-bromoacetic acid methyl ester and 3 g (0.022 mol) of potassiumcarbonate by the same procedure as described in Example 13.

D-p-carbomethoxymethoxyphenylglycine is obtained as its salt bytreatment of the title compound with trifluoroacetic acid as describedin Example 1.

EXAMPLE 257-[D-α-Amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid (Compound No. 07826)

The title compound was obtained from 0.675 g (2.5 mmol) of 7-ADCAt-butyl ester, 0.85 g (2.5 mmol) ofD-N-t-butoxycarbonyl-p-carbomethoxymethoxyphenylglycine and 0.515 g (2.5mmol) of dicyclohexylcarbodiimide by the same procedure as described inExample 6. The initial reaction mixture was stirred at 25° for fivehours.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.19 H.sub.21 N.sub.3 O.sub.7 S . CF.sub.3 COOH                           (549.497)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          45.90          45.70                                               H           4.04           4.11                                               N           7.65           7.61                                               ______________________________________                                    

EXAMPLE 267-[D-α-Amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid (Compound No. 96826)

The title compound was obtained from 1.00 g (2.5 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, 0.85 g (2.5 mmol) ofD-N-t-butoxycarbonyl-p-carbomethoxymethoxyphenylglycine and 0.515 g (2.5mmol) of dicyclohexylcarbodiimide by the same procedure as described inExample 6. The initial reaction mixture was stirred at 25° for twohours.

Analysis of zwitterion: C₂₂ H₂₃ N₅ O₇ S₃ (565.664)

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.22 H.sub.23 N.sub.5 O.sub.7 S.sub.3                       ______________________________________                                                      (565.664)                                                                Theory       Found                                                   ______________________________________                                        C          46.72          46.49                                               H           4.10           4.16                                               N          12.38          12.11                                               ______________________________________                                    

EXAMPLE 277-[D-α-Amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-(1,2,3-triazol-4ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 19736)

The title compound was obtained from 1.55 g (3.56 mmol) of the activatedester of D-N-t-butoxycarbonyl-p-carbomethoxymethoxyphenylglycine, 1.11 g(3.56 mmol) of7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylic acid and0.72 g (7.12 mmol) of triethylamine by the same procedure as describedin Example 8.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.21 H.sub.22 N.sub.6 O.sub.7 S.sub.2 . 1/2 CF.sub.3                      COOH . 1/2 H.sub.2 O                                                          (600.583)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          44.00          44.09                                               H           3.94           4.06                                               N          13.99          13.59                                               ______________________________________                                    

EXAMPLE 287-[D-α-Amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 26436)

The title compound was obtained from 1.00 g (2.6 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, 0.883 g (2.6 mmol) ofD-N-t-butoxycarbonyl-p-carbomethoxymethoxyphenylglycine and 0.536 g (2.6mmol) of dicyclohexylcarbodiimide by a procedure similar to thatdescribed in Example 6. The initial reaction mixture was stirred for 12hours at 25° before work-up. Deblocking was accomplished by stirring amixture of the crude adduct and 2 ml of benzenethiol in 10 ml oftrifluoroacetic acid at 25° for one hour. Concentration of the reactionmixture gave a residue which crystallized upon addition of ether and wasrecrystallized from ethanol-ether (0.500 g).

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.21 H.sub.23 N.sub.7 O.sub.7 S.sub.2 . 1 H.sub.2 O                       (567.619)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          44.44          44.33                                               H           4.44           4.35                                               N          17.27          16.60                                               ______________________________________                                    

EXAMPLE 29D-N-t-Butoxycarbonyl-p-t-butoxycarbonylmethylaminophenylglycine

A mixture of 2.5 g (7.7 mmol) ofD-N-t-butoxycarbonyl-p-aminophenylglycine, 1.8 g (9.2 mmol) ofα-bromoacetic acid t-butyl ester and 2.5 g (19.3 mmol) of diisopropylethylamine in 15 ml of ethanol was stirred at 25° for 48 hours. Thesolvent was removed in vacuo, the residue was diluted with ethyl acetateand sodium bicarbonate and the pH was adjusted to 2.5. The layers wereseparated and the aqueous phase was again extracted with ethyl acetate.The combined extracts were washed with saturated sodium chloridesolution, dried (MgSO₄) and concentrated to give 1.1 g (32%) of thetitle compound.

D-p-carboxymethylaminophenylglycine is obtained as its salt by treatingthe title compound with trifluoroacetic acid as described in Example 1.

EXAMPLE 307-[D-α-Amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-desacetoxycephalosporanicacid (Compound No. 51436)

The title compound was obtained from 0.240 g (1.0 mmol) of 7-ADCAt-butyl ester, 0.380 g (1.0 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylaminophenylglycine and0.210 g (1.0 mmol) of dicyclohexylcarbodiimide in 9:1 ethylacetate-methylene chloride by the same procedure as described in Example6. Before deblocking, the intermediate was purified by chromatography onsilica gel with methylene chloride-ethyl acetate-acetic acid (4:1:.01).Deblocking was accomplished by the procedure described in Example 28.

    ______________________________________                                        Analysis of zwitterion:                                                                     C.sub.18 H.sub.20 N.sub.4 O.sub.6 S . 1.5 H.sub.2 O                           (447.480)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          48.32          48.53                                               H           5.18           4.88                                               N          12.52          12.34                                               ______________________________________                                    

EXAMPLE 317-[D-α-Amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol2-ylthiomethyl)-3-cephem-4-carboxylic acid (Compound No. 48536)

The title compound was obtained from 0.66 g (1.77 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylaminophenylglycine, 0.652 g(1.77 mmol) of7-amino-3-(5-methyl-1,3,4,thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.376 g (1.77 mmol) of dicyclohexylcarbodiimidein 18 ml of ethyl acetate and 2 ml of methylene chloride by the sameprocedure as described in Example 6. The initial reaction mixture wasstirred at 0° for one hour, then at 25° for two hours. Beforedeblocking, the intermediate was purified by chromatography on silicagel with ethyl acetate-benzene. Deblocking was accomplished by theprocedure described in Example 28.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.21 H.sub.22 N.sub.6 O.sub.6 S.sub.3 . CF.sub.3 COOH .                   1(C.sub.2 H.sub.5).sub.2 O                                                    (738.807)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          43.89          43.61                                               H           4.50           4.49                                               N          11.38          11.61                                               ______________________________________                                    

EXAMPLE 32 D-N-t-Butoxycarbonyl-p-carbomethoxymethylaminophenylglycine

Substitution of an equivalent amount of α-bromoacetic acid methyl esterin the procedure of Example 29 for α-bromoacetic acid t-butyl ester,followed by chromatography of the crude product on silica gel with4:1:0.01 methylene chloride-ethyl acetate-acetic acid gave the titlecompound.

D-p-carbomethoxymethylaminophenylglycine is obtained as its salt bytreatment of the title compound with trifluoroacetic acid as describedin Example 1.

EXAMPLE 337-[D-α-Amino-α-(4'-carbomethoxymethylaminophenyl)-acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 96146)

The title compound was obtained from 0.338 g (1 mmol) ofD-N-t-butoxycarbonyl-p-carbomethoxymethylaminophenylglycine, 0.368 g (1mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.212 g (1 mmol) of dicyclohexylcarbodiimide in20 ml of methylene chloride by the same procedure as described inExample 6. The initial reaction mixture was stirred at 0° for 30 minutesthen at 25° for two hours. Before deblocking, the intermediate waschromatographed on silica gel with ethyl acetate-benzene. Deblocking wasaccomplished by stirring a mixture of 0.2 g of7-[D-α-t-butoxycarbonylamino-α-(4'-carbomethoxymethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.5 ml of benzenethiol in 4.5 ml oftrifluoroacetic acid at 0° for 30 minutes, then at 25° for 1.5 hours.The reaction mixture was concentrated and the residue triturated withether to give the title compound (0.2 g) as its salt, which wasrecrystallized from methanol-ether.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.22 H.sub.24 N.sub.6 O.sub.6 S.sub.3 . O.7 CF.sub.3                      CO.sub.2 H                                                                    (644.480)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          43.61          43.42                                               H           3.86           4.23                                               N          13.04          13.34                                               ______________________________________                                    

EXAMPLE 34

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid listed belowis substituted in the procedure of Example 6 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained:

7-amino-3-(1,3,4-thiadiazol-2-ylthiomethyl-3-cephem-4-carboxylic acid.

7-amino-3-(5-ethyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-trifluoromethyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-n-butyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,2,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(3-methyl-1,2,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(3-ethyl-1,2,4-thiadiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(thiazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(2-methylthiazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(4-methylthiazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(2,4-dimethylthiazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,3,4-oxadiazol-2-ylthiomethyl)3-cephem-4-carboxylic acid.

7-amino-3-(5-methyl-1,3,4-oxadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(2-methyloxazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(oxazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(4-methyloxazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(2,4-dimethyloxazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(1-ethyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1-methoxymethyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(4-methyl-1,2,4-triazol-3-ylthiomethyl-3-cephem-4-carboxylicacid.

7-amino-3-(5-methyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1-methyl-1,2,4-triazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1-methyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,5-dimethyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1,3-dimethyl-1,2,4-triazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(4,5-dimethyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-methoxymethyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(4-methyl-5-trifluoromethyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(4-allyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3(5-hydroxy-4-methyl-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(4-ethyl-5-hydroxy-1,2,4-triazol-3-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(1-methyl-1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-methyl-1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(3,5-dimethyl-1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-methylthio-1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(5-methoxymethyl-1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid.

7-amino-3-(4-pyridylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(3-pyridylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(4-pyrimidylthiomethyl)-3-cephem-4-carboxylic acid.

7-amino-3-(2-pyrazinylthiomethyl)-3-cephem-4-carboxylic acid.

EXAMPLE 35

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 10 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carboxyethylthiophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 36

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 12 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carbamylmethylthiophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 37

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 14 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 38

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 21 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 39

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 26 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 40

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 31 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 41

When an equivalent amount of the t-butyl ester of a7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 33 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(4'-carbomethoxymethylaminophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 427-[D-α-Amino-α-(3'-carboxymethylthiophenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.5 g (1.28 mmol) ofD-N-t-butoxycarbonyl-m-t-butoxycarbonyl-methylthiophenylglycine, 0.5 g(1.3 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl-3-cephem-4-carboxylicacid t-butyl ester and 0.265 g (1.3 mmol) of dicyclohexylcarbodiimide bythe same procedure as described in Example 6.

EXAMPLE 43

When an equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid, or a 7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acidlisted in Example 34 is substituted in the procedure of Example 42 for7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(3'-carboxymethylthiophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 447-[D-α-Amino-α-(3'-carboxymethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.66 g (1.77 mmol) ofD-N-t-butoxycarbonyl-m-t-butoxycarbonylmethylaminophenylglycine, 0.652 g(1.77 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.376 g (1.77 mmol) of dicyclohexylcarbodiimideby the same procedure as described in Example 31.

EXAMPLE 45

When an equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid or a 7-amino-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acidlisted in Example 34 is substituted in the procedure of Example 44 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(3'-carboxymethylaminophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid is obtained.

EXAMPLE 467-[α-Amino-α-(3'-carbamylmethoxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.648 g (2.0 mmol) ofD-N-t-butoxycarbonyl-m-carbamylmethoxyphenylglycine, 0.800 g (2.0 mmol)of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.412 g (2.0 mmol) of dicyclohexylcarbodimide bythe same procedure as described in Example 6.

EXAMPLE 47

When an equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid,7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylic acid or a7-amino-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acid listed inExample 34 is substituted in the procedure of Example 46 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(3'-carbamylmethoxyphenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 487-[α-Amino-α-(2'-carboxymethylthiophenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.5 g (1.28 mmol) ofD-N-T-butoxycarbonyl-o-t-butoxycarbonylmethylthiophenylglycine, 0.5 g(1.3 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.265 g (1.3 mmol) of dicyclohexylcarbodiimide bythe same procedure as described in Example 6.

EXAMPLE 49

When a equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid or a 7-amino-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acidlisted in Example 34 is substituted in the procedure of Example 48 for7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(2'-carboxymethylthiophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 507-[α-Amino-α-(2'-carboxymethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.66 g (1.77 mmol) ofD-N-t-butoxycarbonyl-o-t-butoxycarbonylmethylaminophenylglycine, 0.652 g(1.77 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl-3-cephem-4-carboxylicacid t-butyl ester and 0.376 g (1.77 mmol) of dicyclohexylcarbodiimideby the same procedure as described in Example 31.

EXAMPLE 51

When an equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid or a 7-amino-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acidlisted in Example 34 is substituted in the procedure of Example 50 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid t-butyl ester,the appropriate7-[α-amino-α-(2'-carboxymethylaminophenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 527-[α-Amino-α-(2'-carbamylmethoxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid

The title compound is obtained from reaction of 0.648 g (2.0 mmol) ofD-N-t-butoxycarbonyl-o-carbamylmethoxyphenylglycine, 0.800 g (2.0 mmol)of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.412 g (2.0 mmol) of dicyclohexylcarbodiimide bythe same procedure as described in Example 6.

EXAMPLE 53

When an equivalent amount of the t-butyl ester of7-amino-3-desacetoxycephalosporanic acid,7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-amino-3-(1,2,3-triazol-4-ylthiomethyl)-3-cephem-4-carboxylicacid or a 7-amino-3-heterocyclicthiomethyl-3-cephem-4-carboxylic acidlisted in Example 34 is substituted in the procedure of Example 52 for7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester, the appropriate7-[α-amino-α-(2'-carbamylmethoxyphenyl)acetamido]-3-heterocyclicthiomethyl-3-cephem-4-carboxylicacid is obtained.

EXAMPLE 547-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-(1-pyridiniummethyl)-3-cephem-4-carboxylicacid

To a solution of 5.4 g (0.01 mol) of7-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]cephalosporanic acidsodium salt in 25 ml of water is added 2.23 g (0.023 mol) of potassiumthiocyanate and 2 ml (0.028 mol) of pyridine. The reaction mixture isheated at 65°-70° for seven hours. After cooling, the mixture is dilutedwith 100 ml of water and the aqueous solution is chromatographed on acolumn of cross-linked polystyrene polymer (Amberlite XAD-2). Theinorganic salts are eluted with water, then the product is eluted with95% ethanol. Evaporation of the eluent gives the title compound.

EXAMPLE 557-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-methylthiomethyl-3-cephem-4-carboxylicacid

Acylation of 7-amino-3-methylthiomethyl-3-cephem-4-carboxylic acid(Belgian Patent No. 743,754) withD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine accordingto the procedure of Example 2 gives the title compound.

EXAMPLE 567-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-methoxymethyl-3-cephem-4-carboxylicacid

Acylation of 7-amino-3-methoxymethyl-3-cephem-4-carboxylic acid [J. Med.Chem., 14,113(1971)] withD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylthiophenylglycine accordingto the procedure of Example 2 gives the title compound.

EXAMPLE 57

An injectable pharmaceutical composition is formed by adding sterilewater or sterile saline solution (2 ml) to 500 mg of7-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, sodium salt.

Pharmaceutical compositions of the other antibacterial compounds withinthe scope of Formula I or disclosed above may be formulated in a similarmanner.

EXAMPLE 587-[D-α-Amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (Compound No. 76746)

The title compound was obtained from 1.5 g (3.02 mmol) ofD-N-t-butoxycarbonyl-p-t-butoxycarbonylmethylaminophenylglycine, 1.16 g(3.02 mmol) of7-amino-3-(1-methyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.640 g (3.02 mmol) of dicyclohexylcarbodiimidein dry methylene chloride by the procedure described in Example 6. Theinitial reaction mixture was stirred at 0° for one hour then at 25° fortwo hours. Before deblocking, the intermediate was purified bychromatography on silica gel with ethyl acetate-benzene. Deblocking wasaccomplished by the procedure described in Example 28.

    ______________________________________                                        Analysis of salt:                                                                           C.sub.20 H.sub.22 N.sub.8 O.sub.6 S.sub.2 . 1/2 CF.sub.3                      COOH . 3/4 H.sub.2 O                                                          (605.099)                                                       ______________________________________                                                 Theory       Found                                                   ______________________________________                                        C          41.68          41.70                                               H           4.00           4.12                                               N          18.52          18.33                                               ______________________________________                                    

The salt is converted to the zwitterion as in Example 6.

EXAMPLE 59 D-N-t-Butoxycarbonyl-p-carbamylmethylaminophenylglycine

A solution of 2.84 g (0.01 mol) ofD-N-t-butoxycarbonyl-p-aminophenylglycine hydrate, 2.84 g (0.022 mol) ofN,N-diisopropylethylamine and 2.22 g (0.012 mol) of α-iodoacetamide in50 ml of absolute alcohol was refluxed for three hours. The reactionmixture was concentrated in vacuo, the residue was dissolved in waterand the solution brought to pH 8.5 with 5% sodium bicarbonate and washedwith ethyl acetate. More ethyl acetate was added, the pH was adjusted to1.5 with 3N hydrochloric acid and the layers were separated. The organicphase was washed (H₂ O), dried (MgSO₄) and concentrated to give thetitle compound. D-p-carbamylmethylaminophenylglycine is obtained as itssalt from treatment of the title compound with trifluoroacetic acid asdescribed in Example 1.

EXAMPLE 607-[D-α-Amino-α-(4'-carbamylmethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound No. 72546)

To a mixture of 1.00 g (2.5 mmol) of7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.81 g (2.5 mmol) ofD-N-t-butoxycarbonyl-p-carbamylmethylaminophenylglycine in 10 ml ofmethylene chloride and 5 ml of dimethylformamide was added a solution of0.515 g (2.5 mmol) of dicyclohexylcarbodiimide in 10 ml of methylenechloride. The reaction mixture was stirred at 25° for two hours, then itwas filtered and the filtrate was washed with 3N hydrochloric acid, 5%sodium bicarbonate and water. After drying (MgSO₄), the solvent wasremoved in vacuo an the residue was dissolved in a small amount ofchloroform, filtered and chromatographed on silica gel to give7-[D-α-(t-butoxycarbonylamino)-α-(4'-carbamylmethylaminophenyl)acetamido]-3-(5-methyl-1,3,4-thiadazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester. Deblocking was accomplished by stirring a solutionof the intermediate in 10 ml of cold trifluoroacetic acid and 2 ml ofanisole at 25° for two hours as previously described to give the salt ofthe title compound. The zwitterion is obtained from the trifluoroacetatesalt as in Example 6.

I claim:
 1. A compound of the formula: ##STR3## in which: Y is O, NH orS;n is one to five; R is NH₂ or OR', where R' is hydrogen or lower alkylof from one to four carbon atoms; M is hydrogen or an alkali metal orammonium cation; and A is hydrogen, acetoxy or pyridyl.
 2. A compound asclaimed in claim 1, where n is one or two.
 3. A compound as claimed inclaim 2, where R is OH, NH₂ or OCH₃.
 4. A compound as claimed in claim3, where A is H or acetoxy.
 5. A compound as claimed in claim 4, beingthe compound7-[α-amino-α-(4'-carboxymethylthiophenyl)acetamido]-3-desacetoxycephalosporanicacid.
 6. A compound as claimed in claim 4, being the compound7-[α-amino-α-(4'-carboxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 7. A compound as claimed in claim 4 being the compound7-[α-amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 8. A compound as claimed in claim 4 being the compound7-[α-amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 9. A compound as claimed in claim 4 being the compound7-[α-amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-desacetoxycephalosporanicacid.
 10. A compound as claimed in claim 4 being the compound7-[α-amino-α-(4'-carboxymethoxyphenyl)acetamido]-cephalosporanic acid.11. A pharmaceutical composition comprising an antibacterially effectiveamount of a compound as claimed in claim 1 and a pharmaceuticallyacceptable carrier therefor.
 12. The composition of claim 11 comprisingthe compound7-[α-amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 13. The composition of claim 11 comprising the compound7-[α-amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 14. The composition of claim 11 comprising the compound7-[α-amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-desacetoxycephalosporanicacid.
 15. The composition of claim 11 comprising the compound7-[α-amino-α-(4'-carboxymethoxyphenyl)acetamido]-cephalosporanic acid.16. A method of treating bacterial infections comprising administeringby injection to a warm-blooded animal a pharmaceutical compositioncomprising an effective but nontoxic dose of a compound as claimed inclaim 1 and a pharmaceutical carrier therefor.
 17. The method of claim16 in which the compound is7-[α-amino-α-(4'-carbamylmethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 18. The method of claim 16 in which the compound is7-[α-amino-α-(4'-carbomethoxymethoxyphenyl)acetamido]-3-desacetoxycephalosporanicacid.
 19. The method of claim 16 in which the compound is7-[α-amino-α-(4'-carboxymethylaminophenyl)acetamido]-3-desacetoxycephalosporanicacid.
 20. The method of claim 16 in which the compound is7-[α-amino-α-(4'-carboxymethoxyphenyl)acetamido]cephalosporanic acid.